vdsstream VDS - Plasmodium falciparum Adenylosuccinate Synthetase

Target (protein/gene name): Plasmodium falciparum Adenylosuccinate Synthetase (PfAS)
NCBI Gene # or RefSeq#: NCBI Gene #814251
Protein ID (NP or XP #) or Wolbachia#: XP_001350257.1
Organism (including strain): Plasmodium falciparum
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
Disease Information (sort of like the Intro to your Mini Research Write up): Plasmodium falciparum is a protozoan parasite accounting for the majority of the approximately one million worldwide deaths from malaria annually [1]. Because of P. falciparum’s increasing resistance to current drugs such as artemisinins [2], discovering novel drugs for this pathogen is crucial for maintaining effective malaria treatment and reducing mortality.
Link to TDR Targets page (if present): http://www.tdrtargets.org/targets/view?gene_id=2266
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein: P. falciparum adenylosuccinate (PfAS) catalyzes the synthesis of adenylosuccinate from inosine monophosphate (IMP) and aspartate as an intermediate step of the purine salvage pathway. Since P. falciparum cannot synthesize purines through any other means, PfAS is an essential protein for P. falciparum, and disrupting its function would hinder P. falciparum from synthesizing nucleic acids and replicating DNA, impeding its reproduction in the host [3].
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): monomer
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): " In ancillary studies, it was shown for the first time in vivo that the parenteral administration of L-alanosine reduces the specific activity of intratumoral adenylosuccinate synthetase by 70% and depresses the synthesis of DNA to an equivalent or greater extent..." [4]

EC#: 6.3.4.4
Link to BRENDA EC# page
BRENDA enzyme mechanism schematic:
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): BIOMOL® Green free phosphate spectrophotometric assay
link to Sigma (or other company) page for assay (see Sigma links below): BIOMOL® Green
citation to paper that contains assay information: [5]
links to assay reagents (substrates) pages. GTP, IMP, L-aspartate
cost and quantity of substrate reagents, supplier, and catalog #: see links above

Structure (PDB or Homology model)

PDB ID: 1P9B [3]

Current Inhibitors:
Expression Information (has it been expressed in bacterial cells): "Recombinant PfAdSS [PfAS] expressed in E. coli BL21 (DE3) was purified as reported" [3].
Purification Method: Nickel-NTA affinity chromatography
Image of protein (PyMol with features delineated and shown separately):
PfAS: C green, O red, N blueGTP: C pink, O red, N blue, P orange; IMP: C cyan, O red, N blue, P orange; Hadacidin: C yellow, O red, N blue; Mg2+: green sphere
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MNIFDHQIKNVDKGNVVAILGAQWGDEGKGKIIDMLSEYSDITCRFNGGANAGHTISVNDKKYALHLLPCGVLYDNNISVLGNGMVIHVKSLMEEIESVGGKLLDRLYLSNKAHILFDIHQIIDSIQETKKLKEGKQIGTTKRGIGPCYSTKASRIGIRLGTLKNFENFKNMYSKLIDHLMDLYNITEYDKEKELNLFYNYHLKLRDRIVDVISFMNTNLENNKKVLIEGANAAMLDIDFGTYPYVTSSCTTVGGVFSGLGIHHKKLNLVVGVVKSYLTRVGCGPFLTELNNDVGQYLREKGHEYGTTTKRPRRCGWLDIPMLLYVKCINSIDMINLTKLDVLSGLEEILLCVNFKNKKTGELLEKGCYPVEEEISEEYEPVYEKFSGWKEDISTCNEFDELPENAKKYILAIEKYLKTPIVWIGVGPNRKNMIVKKNFNLN
length of your protein in Amino Acids: 442 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 50.0648 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 53915, assuming all pairs of Cys residues form cystines; 53290, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.

results

CDS Gene Sequence (paste as text only): ATGAACATATTTGATCATCAAATAAAAAATGTGGATAAAGGGAATGTAGTTGCAATATTAGGTGCACAATGGGGTGATGAAGGGAAAGGAAAAATAATTGATATGTTATCAGAATATTCTGATATTACTTGTAGATTTAATGGAGGTGCTAATGCAGGACATACGATATCAGTAAATGATAAGAAATATGCTTTACATTTATTACCATGTGGTGTATTATATGATAATAATATAAGTGTATTAGGAAACGGAATGGTAATACATGTAAAATCATTAATGGAAGAAATTGAATCAGTTGGGGGAAAGTTGTTAGATAGATTATATTTATCAAATAAAGCACATATATTATTTGATATTCATCAAATTATTGATTCAATCCAAGAAACGAAAAAATTAAAAGAAGGAAAACAAATAGGTACAACAAAAAGAGGTATTGGACCATGTTATTCTACTAAAGCTTCCAGAATAGGTATAAGATTAGGAACTTTAAAAAATTTTGAAAATTTTAAAAATATGTATAGTAAATTAATAGACCACTTAATGGATTTATATAATATAACAGAATATGACAAAGAAAAAGAACTCAACTTATTTTATAATTATCACTTAAAGTTAAGAGATAGAATAGTTGATGTTATTTCCTTTATGAATACAAATTTAGAAAACAACAAAAAAGTATTAATTGAAGGTGCTAATGCAGCTATGTTAGATATTGATTTTGGAACATATCCATATGTAACTAGTAGCTGTACAACAGTTGGTGGGGTTTTCTCAGGACTTGGAATTCATCATAAAAAACTGAATTTAGTTGTAGGTGTAGTTAAAAGTTACTTAACCAGAGTTGGATGTGGCCCTTTCTTAACTGAATTAAATAATGACGTTGGTCAATATTTAAGAGAAAAAGGTCATGAATATGGAACGACTACCAAGAGACCAAGAAGGTGTGGATGGCTCGACATACCAATGTTATTATATGTTAAGTGCATTAATAGTATTGATATGATAAACTTAACAAAATTGGATGTTTTATCTGGATTAGAGGAAATATTATTGTGTGTCAATTTTAAAAATAAAAAAACAGGTATCAATTAAAGCATATGTATATTTTATATGTGTAATATGTAAATATTATATCTTAATGTTTTCATATATATATATATATATATATATATACATTTATTTATTTATTTTTTTTTTTTTTTAGGAGAACTGCTTGAAAAGGGTTGCTACCCTGTTGAAGAAGAAATATCAGAAGAATATGAACCTGTTTATGAAAAATTCAGTGGATGGAAAGAAGACATCTCAACTTGTAATGAATTTGATGAATTACCAGAAAATGCAAAAAAATATATTTTAGCTATAGAGAAATATTTAAAAACTCCAATAGTTTGGATTGGTGTAGGTCCTAATAGAAAAAATATGATAGTTAAAAAGAATTTTAACCTAAACTAA
GC% Content for gene: 26.4%
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
ATGAACATCTTCGACCATCAAATTAAGAATGTTGACAAAGGTAACGTGGTAGCGATCCTG GGTGCGCAGTGGGGTGACGAGGGTAAAGGCAAAATCATTGACATGCTGTCTGAATACTCT GACATCACTTGCCGTTTCAACGGTGGCGCGAATGCGGGTCACACCATCTCTGTTAACGAT AAGAAATACGCGCTGCACCTGCTGCCTTGCGGTGTTCTGTACGACAACAACATCTCCGTT CTCGGCAATGGTATGGTTATTCACGTTAAGTCTCTCATGGAGGAAATCGAGTCTGTTGGC GGTAAACTGCTCGACCGCCTGTACCTGTCTAACAAAGCGCACATCCTGTTCGACATCCAC CAGATCATCGATTCTATCCAGGAAACCAAAAAGCTGAAGGAAGGTAAACAAATCGGTACG ACCAAGCGTGGTATCGGCCCGTGCTACTCTACCAAAGCGTCTCGTATTGGTATCCGCCTG GGCACCCTCAAGAACTTTGAAAACTTCAAAAACATGTACTCTAAGCTGATTGACCACCTG ATGGACCTGTACAATATCACCGAGTATGACAAGGAAAAGGAGCTCAACCTCTTCTACAAC TATCACCTGAAGCTGCGTGACCGTATCGTTGACGTTATCTCTTTTATGAACACCAACCTG GAGAACAACAAGAAGGTACTGATCGAAGGTGCGAACGCGGCCATGCTCGATATTGACTTC GGTACCTACCCGTACGTGACGTCTTCTTGCACTACCGTTGGTGGTGTTTTCTCTGGTCTC GGTATCCATCACAAGAAACTGAACCTCGTTGTAGGTGTAGTCAAATCTTACCTGACCCGT GTTGGTTGCGGCCCATTCCTGACCGAACTGAATAATGACGTTGGTCAGTATCTCCGCGAG AAAGGTCACGAATACGGTACTACTACGAAACGTCCGCGTCGTTGTGGTTGGCTGGATATT CCGATGCTGCTGTACGTTAAATGCATCAACTCTATCGACATGATCAACCTGACGAAACTG GACGTTCTGTCCGGTCTCGAAGAGATTCTGCTCTGTGTTAACTTTAAGAACAAAAAAACC GGTGAACTCCTCGAAAAAGGCTGTTACCCAGTTGAGGAAGAAATCTCTGAAGAATACGAA CCTGTTTACGAGAAATTCAGCGGTTGGAAGGAGGACATCTCTACCTGCAATGAGTTCGAC GAGCTCCCTGAGAACGCGAAGAAGTACATTCTGGCAATCGAAAAGTACCTCAAAACCCCG ATCGTTTGGATCGGTGTAGGTCCGAACCGTAAGAATATGATCGTAAAAAAGAATTTCAAT CTGAACTAA
GC% Content for gene (codon optimized): 47.1%

Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): see link below
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.

Primer design results for 'tail' primers (this is just 2 sequences):

References:

Murray, C. J. et al., Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet 2012, 379 (9814), 413-31.
Takala-Harrison, S. et al., Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis 2015, 211 (5), 670-9.
Eaazhisai, K. et al., Crystal structure of fully ligated adenylosuccinate synthetase from Plasmodium falciparum. J Mol Biol 2004, 335 (5), 1251-64.
Tyagi, A. K.; Cooney, D. A., Identification of the antimetabolite of L-alanosine, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, in tumors and assessment of its inhibition of adenylosuccinate synthetase. Cancer Res 1980, 40 (12), 4390-7.
Myers, C. L. et al., Characterization of wall teichoic acid degradation by the bacteriophage φ29 appendage protein GP12 using synthetic substrate analogs. Journal of Biological Chemistry: 2015; p 5.